confirming apoptosis - (Feb/12/2010 )

i want to detect, if cells are going into apoptosis. So i wondered which practical competences are these days used?
I dont have any experience with this field,.
Can anyone help me?

susan

-susanna-

susanna on Feb 12 2010, 10:55 AM said:

Hi,

i want to detect, if cells are going into apoptosis. So i wondered which practical competences are these days used?
I dont have any experience with this field,.
Can anyone help me?

susan

You can show apoptosis in several different ways. By showing activation of key-proteins in the signal-pathways (eg Caspase-3 via IF or FACS), showing fractionation of DNA (classical by isolating DNA and running it on a gel, by TUNEL assay) or by detecting Annexin V (IF). These are just a couple of examples of available assays. Most of these are readily available as kits from several vendors.
It would be a wise idea first do to a literature search first. To see what works for your cells your working with, experimental design etc.
So do some reading and post if you have some more specific questions or ideas what you exactly want to do.

-snoopyx-

snoopyx on Feb 12 2010, 09:09 AM said:

susanna on Feb 12 2010, 10:55 AM said:

Hi,

i want to detect, if cells are going into apoptosis. So i wondered which practical competences are these days used?
I dont have any experience with this field,.
Can anyone help me?

susan

You can show apoptosis in several different ways. By showing activation of key-proteins in the signal-pathways (eg Caspase-3 via IF or FACS), showing fractionation of DNA (classical by isolating DNA and running it on a gel, by TUNEL assay) or by detecting Annexin V (IF). These are just a couple of examples of available assays. Most of these are readily available as kits from several vendors.
It would be a wise idea first do to a literature search first. To see what works for your cells your working with, experimental design etc.
So do some reading and post if you have some more specific questions or ideas what you exactly want to do.

actually i want to confirm, if tumorcells are still "alive", after radiation therapy . And this has to be done, in patients.
I already did some reading. And i know which possibilities there are, but this is just in theory.
Do you have an idea, which technic could be applicable for this purpose?

-susanna-

susanna on Feb 16 2010, 07:39 AM said:

actually i want to confirm, if tumorcells are still "alive", after radiation therapy . And this has to be done, in patients.
I already did some reading. And i know which possibilities there are, but this is just in theory.
Do you have an idea, which technic could be applicable for this purpose?

If I understand you right your plan is to exclude the dead cells, undergoing apoptosis to count the live cells? All my experience for now is based on in vitro stuff in cell culture. Never tried to show apoptosis in vivo. The only thing I can think off is to stain biopsy-material.
I don't know to much about cancer. But in the case of "Blood" cancer you could try to draw blood, isolate the cells you desire and run it through FACS for spec. markers (double stain: marker for cancer cells + marker for apoptosis). If you want some quantitative numbers the FACS would be the best thing. From biopsy material you could make cell suspensions, but maybe this is to invasive already.